The studies in progress are designed to explore the mechanism by which AML serum exposed to Staphylococcal protein A (PA) becomes cytotoxic to leukemic cells. Acute myelogenous leukemia (AML) sera, when exposed to killed Staphylococcus aureus, Cowan I (SAC) kill AML blasts in culture. Addition of an equal volume (15%) of untreated AML sera to such cultures partially blocks this effect. Utilizing sepharose-bound PA, we purified leukemic IgG and resuspended it to physiological concentration (900 mg/dl). In 13 experiments, SAC-treated AML sera mediated marked cytotoxicity against AML blasts (40.6 plus or minus 20.1% viable cells compared to control). In simultaneous cultures, the addition of AML IgG to these same cultures resulted in 65.9 plus or minus 24.4% viable cells, indicating that AML IgG demonstrates similar "blocking" ability to whole AML serum. Since SAC removes IgG and, therefore, reduces protein content, we explored the need for protein in 24-hour cultures of AML blasts. We found that blasts cultured with no protein in the media were 51.1% viable (compared to control) while the addition of SAC-treated serum to cultures resulted in 50.8% viable cells (n=12). With purified AML IgG as the only protein source, viability was somewhat enhanced (66.9%). Additionally, when IgG was added to untreated autologous AML sera, the number of viable cells was 108.8% of control cultures. Thus, AML IgG partially blocks SAC-induced cytotoxicity, but does not appear to act as a growth factor nor does it mediate cytotoxicity. Studies using trypsin-treated sera further confirm the presence of a cytotoxic factor. SAC-treated sera reduced viable AML blasts to 46.7 plus or minus 18.1%. When the same SAC-treated sera were exposed to insoluble trypsin, centrifuged to remove trypsin and cultured with leukemic blasts, no cytotoxicity was seen (97.0 plus or minus 8.2% viable cells). The same sera, trypsin-treated but not SAC-treated, also displayed no cytotoxic or viability-potentiating effects (95.7 plus or minus 24.0%). SAC-treated AML sera were filtered through Amicon filters of varying pore sizes. In 11 experiments, the greater than 300,000 molecular weight (MW) fraction showed no activity, while the cytotoxic factor was concentrated in the less than 100,000 MW fraction (38.5 plus or minus 25.2% of control), but not in the less than 50,000 MW fraction. Thus, the cytotoxic factor appears to be a trypsin-sensitive protein in the 50,000 to 100,000 MW range. A possible explanation for these data would be elution of PA from SAC with direct toxicity. However, addition of soluble PA directly to cultures resulted in no cytotoxicity.